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Department of Physiology and Morphology, Institute of Medicinal Chemistry, Hoshi University, Shinagawa-ku, Tokyo 142-8501, Japan
Submitted 5 November 2002 ; accepted in final form 19 March 2003
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-glycyrrhetinic acid (18-GA), an inhibitor of
gap junctions, and enhanced by IBMX, a cAMP-phosphodiesterase (PDE) inhibitor.
These enhanced EDHF-type responses were very similar in magnitude between
diabetic and age-matched control rats. The EDHF-type relaxation was enhanced
by cilostamide, a PDE3-selective inhibitor, but not by Ro 20–1724, a
PDE4-selective inhibitor. The expression levels of the mRNAs and proteins for
two cAMP PDEs (PDE3A, PDE3B) were significantly increased in STZ-induced
diabetic rats, but those for PDE4D were not. We conclude that the impairment
of EDHF-type relaxations in STZ-induced diabetic rats may be attributed to a
reduction in the action of cAMP via increased PDE activity.
endothelium-derived hyperpolarizing factor; adenosine 3',5'-cyclic monophosphate; streptozotocin
Although the identity of EDHF is still controversial
(46), there is evidence that
EDHF-type relaxations involve the transfer of a mediator from the endothelium
to the smooth muscle via myoendothelial gap junctions
(6,
20,
59). Indeed, EDHF-type
responses are attenuated by connexin-mimetic peptides
(6,
17,
30) and by 18- and
18
-glycyrrhetinic acids (GA), aglycones that disrupt gap junction
plaques at points of cell-to-cell contact
(12,
66,
69).
It was recently reported that cAMP facilitates EDHF-type relaxation in
conduit arteries by enhancing electrotonic conduction via gap junctions
(26). In fact, EDHF-type
relaxations are potentiated when cAMP hydrolysis is inhibited by the
phosphodiesterase (PDE) inhibitor IBMX but remain susceptible to a combination
of apamin plus charybdotoxin
(65). In accord with such a
role for gap junctions, the EDHF-type relaxations and cAMP accumulation evoked
by ACh are inhibited by synthetic connexin-mimetic peptides, which interrupt
intercellular communication in a connexin-specific fashion, and by
18-GA (7,
27,
65).
The intracellular level of cAMP is dynamically regulated by the concerted actions of adenylyl cyclases and cyclic nucleotide PDE. To date, at least 11 distinct PDE families have been identified, in total containing more than 50 different PDE enzyme variants each encoded by several genes (3, 43, 62). In several animal species, an examination of the profiles of cAMP PDE activities in vascular smooth muscle cells (VSMCs) in contractile arteries identified PDE3 and PDE4 family members as being primarily responsible for cAMP PDE activity in these vessels (54). PDE3 is a high-affinity PDE (low-Km cAMP PDE) that is sometimes referred to as cGMP-inhibited PDE (3, 44). Specific PDE3 inhibitors (e.g., cilostamide, milrinone, and lixazinone) promote smooth muscle relaxation, stimulate myocardial contractility, and inhibit platelet aggregation, suggesting an involvement of PDE3 in the regulation of these physiological processes (13, 28, 43). Molecular cloning studies revealed the presence of two distinct PDE3s, an adipocyte type (PDE3B) and a cardiac type (PDE3A) (14, 45, 61, 63). PDE3A mRNA is abundantly expressed in heart and vascular smooth muscle, whereas PDE3B mRNA is expressed in white and brown adipose tissue, hepatocytes, spermatocytes, and embryonic neuroepithelium, including the neural retina (58, 63). This tissue-specific distribution of PDE3A mRNA suggests the potential for PDE3A to be involved in diabetic vascular disease (48). Four PDE4 genes have been identified (PDE4A, PDE4B, PDE4C, and PDE4D), and PDE4 activity is detectable in most mammalian cells except blood platelets (11, 28). Perhaps because PDE4 inhibitors induce only weak vasorelaxations (54), the expression of PDE4s in blood vessels has not been studied extensively. Interestingly, it was reported recently that two PDE4D "long forms" (PDE4D3 and PDE4D5) are expressed in rat and human VSMCs (41, 51).
Diabetes mellitus is associated with vascular complications, including an impairment of vascular responsiveness to neurotransmitters in the macro- and microvasculature (15). In the former, there is an accumulating body of evidence to show that the relaxation responses induced in aortic strips by endothelium-dependent agents are weaker in streptozotocin (STZ)-induced diabetic rats than in nondiabetic control rats (10, 34, 37, 50, 53, 55). Recently, several reports indicated that an impairment of endothelium-dependent hyperpolarization and/or relaxation in the microvasculature can be induced by ACh in diabetic rats (23, 42, 68).
For the present study, we designed experiments to investigate the mechanisms underlying the diabetesrelated impairment of ACh-induced endothelium-dependent vasodilation in the rat superior mesenteric artery. We were especially interested in cAMP-induced modulation of the EDHF-type relaxation in diabetes. We also asked whether mesenteric arteries from control and established diabetic rats might differ in their PDE expression profiles.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Male Wistar rats (8 wk old, 180- to 250-g body wt) received a single injection via the tail vein of 65 mg/kg STZ dissolved in a citrate buffer. Age-matched control rats were injected with the buffer alone. Food and water were given ad libitum. This study was conducted in accordance with the Guide for the Care and Use of Laboratory Animals adopted by the Committee on the Care and Use of Laboratory Animals of Hoshi University (which is accredited by the Ministry of Education, Science, Sports and Culture, Japan).
Measurement of Plasma Glucose
Twelve weeks after the injection of STZ or buffer, plasma glucose was determined with a commercially available enzyme kit (Wako Chemical, Osaka, Japan).
Measurement of Isometric Force
Rats were anesthetized with diethyl ether and euthanized by decapitation 12
wk after treatment with STZ or buffer. The superior mesenteric artery was
rapidly removed and immersed in oxygenated, modified Krebs-Henseleit solution
(KHS). This solution consisted of (in mM) 118.0 NaCl, 4.7 KCl, 25.0
NaHCO3, 1.8 CaCl2, 1.2 NaH2PO4,
1.2 MgSO4, and 11.0 dextrose. The artery was carefully cleaned of
all fat and connective tissue, and ring segments 2 mm in length were
separately suspended by a pair of stainless steel pins in a well-oxygenated
(95% O2-5% CO2) bath of 10 ml of KHS at 37°C. The
rings were stretched until an optimal resting tension of 1.0 g was loaded and
then allowed to equilibrate for at least 60 min. Force generation was
monitored by means of an isometric transducer (model TB-611T; Nihon Kohden).
The tissues were equilibrated for 40 min in the presence of 100 µM
NG-nitro-L-arginine (L-NNA) and 10
µM indomethacin to block nitric oxide synthase and cyclooxygenase,
respectively, before administration of phenylephrine (1 µM). Once the
phenylephrine-induced contraction was established, concentration-response
curves were constructed for the relaxation induced by ACh or sodium
nitroprusside (SNP). Such concentration-response curves were also generated in
the combined presence of L-NNA (100 µM), indomethacin (10
µM), and one of 18-GA, (100 µM for 40 min), IBMX (20 µM for 40
min), cilostamide (1 µM for 40 min), or Ro 20–1724 (10 µM for 40
min). In the experiments with IBMX and cilostamide, an equieffective
concentration of phenylephrine was used (1–3 µM).
Enzyme Immunoassay for cAMP
Mesenteric rings from diabetic and age-matched control rats were incubated for 40 min at 37°C in oxygenated KHS containing L-NNA (100 µM) plus indomethacin (10 µM) with, in some experiments, cilostamide (1 µM) or Ro 20–1724 (10 µM). Phenylephrine (1 µM) was added 5 min before ACh stimulation. Rings were frozen in liquid N2 after the addition of ACh (3 µM) and stored at -80°C. cAMP was subsequently extracted in 6% trichloroacetic acid, followed by neutralization with water-saturated diethyl ether and enzyme immunoassay (Amersham Biosciences UK).
Measurement of Expression of mRNAs for PDEs
Oligonucleotides. The primers used are summarized in Table 1.
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RNA isolation and RT-PCR. RNA was isolated by the guanidinium method (9). Briefly, rat superior mesenteric arteries were carefully isolated and then cleaned of fat and connective tissue. The arteries were homogenized in RNA buffer, and the RNA was quantified by ultraviolet absorbance spectrophotometry. For the RT-PCR analysis, first-strand cDNA was synthesized from total RNA with oligo(dT) 12–18 and a cDNA synthesis kit (Life Sciences). Twenty (GAPDH), thirty (PDE3B), or twenty-seven (PDE4D) PCR cycles (94°C for 1 min, 54°C for 1 min, 72°C for 1 min) or twenty-six (PDE3A) PCR cycles (94°C for 1 min, 56°C for 1 min, 72°C for 1 min) were performed with one-half of the reverse transcription mixture. The PCR products so obtained were analyzed on ethidium bromide-stained agarose (1.5%) gels. The PCR products were quantified by scanning densitometry, with the amount of each product being normalized with respect to the amount of GAPDH product.
Immunoprecipitation and Western Blotting
Snap-frozen mesenteric arterial tissues were homogenized in ice-cold lysis buffer containing 50 mM Tris · HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, and a protease-inhibitor cocktail. The protein concentration was determined by means of a bicinchoninic acid protein assay reagent kit (Pierce). Aliquot samples (200 µg) of tissue homogenates obtained from diabetic and age-matched control rats were then incubated with anti-PDE3A or anti-PDE3B antibody (Santa Cruz Biotechnology, Santa Cruz, CA; 1:100; 4°C, 4 h), followed by the addition of protein G Sepharose (Amersham Biosciences) for 2 h at 4°C. Immunoprecipitates were collected by centrifugation (13,000 g; 1 min), washed three times with buffer containing 50 mM Tris · HCl (pH 7.5), 150 mM NaCl, 0.1% Triton X-100, and a protease inhibitor cocktail, and resuspended in Laemmli's buffer containing mercaptoethanol. Samples were resolved by electrophoresis on 7.5% SDS-PAGE gels and transferred onto polyvinylidene difluoride membranes. Briefly, after the residual protein sites on the membrane were blocked with Block ace (Dainippon-pharm, Osaka, Japan), the membrane was incubated with anti-PDE3A or anti-PDE3B antibody (1:3,000) in blocking solution. Horseradish peroxidase-conjugated anti-goat antibody (Vector) was used at a 1:10,000 dilution in Tween-PBS, followed by detection with a SuperSignal (Pierce).
Drugs
Streptozotocin, phenylephrine, indomethacin, L-NNA, IBMX,
18-GA, SNP, and protease inhibitor cocktail were all purchased from
Sigma (St. Louis, MO). ACh chloride was from Daiichi Pharmaceuticals (Tokyo,
Japan), and cilostamide and Ro 20–1724 were from Calbiochem-Novabiochem
(La Jolla, CA) All drugs were dissolved in saline, except where otherwise
noted. IBMX, 18-GA, cilostamide, and Ro 20–1724 were dissolved in
DMSO; control experiments confirmed the absence of significant effects on
constrictor tone and ACh-induced relaxations at the final vehicle
concentration used. All concentrations are expressed as the final molar
concentration of the base in the organ bath.
Statistical Analysis
Data are expressed as means ± SE. When appropriate, statistical differences were assessed by Dunnett's test for multiple comparisons after a one-way ANOVA; a probability level of P < 0.05 was regarded as significant. Statistical comparisons between concentration-response curves were made by a two-way ANOVA, with Bonferroni's correction for multiple comparisons being performed post hoc (P < 0.05 again being considered significant).
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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At the time of the experiment, all STZ-treated rats exhibited hyperglycemia, their blood glucose concentrations (610.6 ± 29.6 mg/dl; n = 12) being significantly higher than those of the age-matched nondiabetic control rats (124.4 ± 3.4 mg/dl; n = 12). The body weights of the diabetic rats (236.3 ± 4.2 g; n = 13) were significantly lower than those of the age-matched control rats (472.6 ± 3.8 g; n = 13) at the time of the experiment.
ACh-Induced EDHF-Type Relaxation
To investigate the EDHF-type relaxation evoked by ACh in rat mesenteric artery, we performed a series of experiments in which ACh (10-9–10-5 M) was added cumulatively to rings precontracted by phenylephrine (1 µM) in the presence of 100 µM L-NNA plus 10 µM indomethacin. The tension developed in response to 1 µM phenylephrine was 2.15 ± 0.86 g in diabetic mesenteric rings (n = 13) and 2.38 ± 0.7 g in those from the age-matched control rats (n = 13; no significant difference). The concentration-response curves for the peak amplitude of the EDHF-type relaxation induced by ACh in diabetic and age-matched control rats are shown in Fig. 1. The peak relaxation was significantly weaker in mesenteric arteries from diabetic rats [43.9 ± 3.9% and 69.3 ± 3.9% of the phenylephrine-induced tone in diabetic (n = 13) and age-matched control (n = 13) rats, respectively (P < 0.001)]. The EC50 values for the ACh-induced EDHF-type relaxations were 426.1 ± 99.0 and 65.1 ± 7.0 nM in diabetic and age-matched control rats, respectively (P < 0.001). Preparations incubated with 30 mM K+ or denuded of their endothelium were essentially devoid of vasorelaxant activity (data not shown).
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Effect of 18-GA on ACh-Induced EDHF-Type Relaxation
In several previous reports, gap junctional communication was said to
contribute to the NO- and prostanoid-independent relaxations mediated via the
endothelium (6,
12,
17,
20,
30,
66). To examine the part
played by gap junctional communication in the present EDHF-type relaxation,
rings were incubated with 18-GA
(12,
66,
69), a gap junction inhibitor
(as well as with L-NNA + indomethacin) for 40 min before
administration of phenylephrine. As shown in
Fig. 2, ACh
(10-9–10-5 M) caused a reduced
concentration-dependent relaxation in these rings whether they were from
age-matched control rats or diabetic rats. This reduction was significant both
for the age-matched control rats [Fig.
2A, maximal relaxation (Rmax) = 16.3 ±
3.3, n = 6] and for the diabetic rats
(Fig. 2B,
Rmax = 12.2 ± 2.0, n = 9). The present study
confirmed that 18-GA causes no significant alteration in SNP-induced
relaxation (Fig. 2C)
and has no significant effect on phenylephrine-induced contraction (data not
shown).
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Effect of PDE Inhibitors on ACh-Induced EDHF-Type Relaxation
Because PDE inhibitors have been shown to enhance the EDHF-type relaxation to ACh (7, 26, 65), we examined the effects of IBMX treatment on the present EDHF-type relaxation in diabetic and age-matched control rats. As IBMX depressed contraction, the concentration of phenylephrine used in experiments involving this agent was increased to between 1 and 3 µM. The tension developed in response to 1–3 µM phenylephrine in the presence of IBMX was 1.58 ± 0.05 g in diabetic mesenteric rings (n = 8) and 1.47 ± 0.06 g in age-matched control mesenteric rings (n = 8; no significant difference). The concentration-response curves for the peak amplitude of the EDHF-type relaxation induced by ACh in rings pretreated with IBMX are shown in Fig. 2. After incubation with 20 µM IBMX for 40 min, the Rmax shown by rings from age-matched control rats was increased to 90.9 ± 2.5% with a leftward shift in the EC50 value to 52.0 ± 10.4 nM (Fig. 2A; n = 8) whereas in rings from diabetic rats the maximal relaxation was 91.7 ± 1.9% with a leftward shift in the EC50 value to 43.9 ± 9.4 nM (Fig. 2B; n = 8). There was no significant difference in the facilitated EDHF-type relaxation between diabetic and age-matched control rats.
Of the PDE families identified in VSMCs, members of the PDE3 (cGMP inhibited) and PDE4 (cAMP specific) families have been shown to contribute to the regulation of cAMP signaling and its impact on VSMC function (18, 51, 54). To investigate the effects of PDE-specific inhibitors on the present EDHF-type relaxation, we used cilostamide, a specific inhibitor of PDE3, and Ro 20–1724, a specific inhibitor of PDE4. When 1 µM cilostamide was added to rings (together with the usual L-NNA + indomethacin) for 40 min before administration of phenylephrine, there was no significant difference in the EDHF-type relaxation between rings with cilostamide (Rmax 85.4 ± 2.2%, EC50 38.8 ± 5.7 nM; n = 6) and without (Rmax 72.5 ± 4.4%, EC50 61.8 ± 4.8 nM; n = 7) in age-matched control rats (Fig. 3A). However, the EDHF-type relaxation was significantly enhanced between rings with cilostamide (Rmax 82.1 ± 2.1%, EC50 55.2 ± 9.9 nM; n = 11) and without (Rmax 52.4 ± 5.5%, EC50 202.2 ± 48.8 nM; n = 12) in diabetic rats (Fig. 3B). In contrast, 10 µM Ro 20–1724 did not significantly alter the EDHF-type relaxation in rings from either diabetic (Rmax 40.7 ± 8.6%, EC50 137.8 ± 30.4 nM; n = 11) or age-matched control (Rmax 70.8 ± 8.6%, EC50 55.3 ± 4.3 nM; n = 7) rats.
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cAMP Accumulation
cAMP levels were measured in rat mesenteric arterial rings treated with 100 µM L-NNA plus 10 µM indomethacin for 40 min (Fig. 4). Under our conditions, basal cAMP levels were not significantly different between diabetic and age-matched control rats. An ACh (3 µM)-induced cAMP accumulation was evident in both groups at 15 s, and these elevated cAMP levels were rapidly reduced (see 60 s ACh in Fig. 4). The extent of this decrease in cAMP levels (cAMP content at 15 s minus that at 60 s) was significantly larger in diabetic rats (4.868 ± 0.321 pmol/mg protein) than in control rats (2.274 ± 0.386 pmol/mg protein) (P < 0.01). Cilostamide (1 µM), but not Ro 20–1724 (10 µM), caused equivalent sustained increases in cAMP levels at 60 s in control and diabetic groups.
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Expression of mRNAs and Proteins for PDEs
In vascular smooth muscle cells in several species, PDE3 and PDE4 family members are primarily responsible for cAMP-PDE activity in contractile arteries (54). As described above, PDE3 inhibition enhanced the EDHF-type relaxation to ACh in the diabetic mesenteric artery. To investigate the possible mechanisms underlying the impaired ACh-induced EDHF-type relaxation seen in STZ-induced diabetic rats, we examined whether the expressions of the mRNAs and proteins for PDEs might be altered in the diabetic state. Using RT-PCR analysis on the total RNA isolated from superior mesenteric arteries from age-matched control and STZ-induced diabetic rats, we found that the expression of GAPDH mRNA showed no difference between the diabetic and age-matched control groups but the expressions of the mRNAs for PDE3A and PDE3B were significantly increased in the diabetic rats (compared with age-matched control rats). The expression of the mRNA for PDE4D, however, was not significantly increased (Fig. 5). Immunoblots of mesenteric arteries taken from diabetic and age-matched control rats and then treated with anti-PDE3A or anti-PDE3B antibody allowed detection of immunoreactive proteins with molecular masses of 120 and 135 kDa, respectively (Fig. 5C). PDE3A and PDE3B protein levels were increased in the diabetic rats compared with the age-matched control rats.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Impaired endothelium-dependent relaxations have been consistently demonstrated in blood vessels from STZ-induced diabetic rats (15, 33, 34, 36, 37, 50). Although most studies of endothelial dysfunction in diabetes have focused on NO, an impairment of NO- and prostacyclin-resistant relaxation was recently reported in mesenteric arteries from diabetic rats (23, 42, 68). In the present study, our first finding was that the EDHF-type relaxation induced in the mesenteric artery by ACh was attenuated in STZ-induced diabetic rats (Fig. 1).
The identity of EDHF remains controversial
(46). Recently, K+
has been proposed as a candidate for EDHF in rat arteries
(19). The postulated sequence
would be that when ACh binds to muscarinic receptors on the endothelium,
charybdotoxin- and apamin-sensitive K+ channels in the endothelium
are opened and K+ efflux occurs in the myoendothelial space. The
resulting increase in the myoendothelial K+ concentration
hyperpolarizes and relaxes adjacent smooth muscle cells by activating
Ba2+-sensitive K+ channels and ouabain-sensitive
Na+-K+-ATPase. However, there are other candidates for
EDHF, namely endocannabinoids, anandamide, and epoxyeicosatrienoic acids,
which are cytochrome P-450-monooxygenase-derived metabolites of
arachidonic acid (5,
22,
29,
46,
56). Furthermore, recent
evidence suggests that direct heterocellular gap junctional communication
between endothelial and smooth muscle cells may contribute to NO-independent
relaxation (6,
20,
30,
66,
69). In the present study, we
demonstrated that in the rat superior mesenteric artery, ACh-induced EDHF-type
relaxation is mediated via gap junctions (because the relaxation was largely
blocked by pretreatment with 18-GA;
Fig. 2).
Gap junctional communication plays an important role in vascular tissue homeostasis. The hallmark of the vasorelaxation attributed to EDHF is that it is accompanied by membrane hyperpolarization. The hyperpolarization generated in endothelial cells is capable of spreading electrotonically to the underlying smooth muscle cells, most likely via myoendothelial gap junctions. Evidence supporting the transfer of a small molecule (e.g., cAMP) via myoendothelial gap junctions has also been presented. Recently, in culture experiments, it was reported that high glucose reduced gap junction activity (38, 60). Although our data demonstrated that the present EDHF-type relaxation was mediated by gap junctions, it remains to be determined in future studies whether gap junction activity is directly impaired in STZ-induced diabetic rats.
Recent studies reported that cAMP facilitates EDHF-type relaxations in
conduit arteries via gap junctional communication
(7,
26,
27,
65). Experiments with the P
site agonist 2',3'-dideoxyadenosine (2',3'-DDA) and
forskolin, which respectively inhibit and stimulate adenylate cyclase
(16,
32,
39), also provided insights
into the cellular mechanisms that underlie the EDHF phenomenon by
demonstrating a central role for cAMP. In our previous studies
(35) we observed an
NO-independent decrease in the forskolin-induced relaxation after endothelial
denudation in the rat mesenteric bed, and others
(40) reported that the
IC50 value for the cAMP accumulation induced by this agent is
shifted approximately threefold to the right after endothelial denudation in
rabbit arteries. It was also demonstrated that the endothelium is a major
source of cAMP. For example, in the perfused rat mesentery the extracellular
release of cAMP in response to ACh or the Ca2+-ATPase inhibitor
cyclopiazonic acid, both of which evoke EDHF-type relaxations sensitive to Gap
27 peptide and 18-GA (6,
17,
30,
66), is markedly reduced by
endothelial denudation (1,
35). Having found that the
EDHF-type relaxations in our preparations were mediated by gap junctions, we
examined the effects of cAMP PDE inhibitors on EDHF-type relaxation
(Fig. 2). This relaxation in
the rat superior mesenteric artery was potentiated in the presence of IBMX,
and this potentiation was larger in diabetic rats than in control rats (with
the result that in IBMX-treated rings, the EDHF-type relaxations were very
similar between control and diabetic rats). These findings suggest that cAMP
signaling is impaired in the diabetic state. Indeed, in our previous study
(2), the ACh-induced cAMP
release from a mesentery preparation was decreased in the diabetic rat
group.
cAMP signaling in mammalian cells is terminated by cyclic nucleotide PDEs, a multifamily class of enzymes that catalyze the hydrolysis of cyclic nucleotides to 5'-nucleotide monophosphates (which do not activate cAMP effector proteins; Refs. 3, 4). Among the various PDEs, PDE3 and PDE4 are of most interest because they preferentially hydrolyze cAMP (54). It was reported recently that PDE3 and PDE4 activities are altered in diabetes (48, 49). Nagaoka et al. (48) reported that the increased PDE3 activity in the aorta of atherosclerosis-prone insulin-resistant cp/cp rats correlated positively with an increased amount of PDE3A mRNA. Also, Tang et al. (64) reported that in adipose tissue of obese insulin-resistant diabetic KKAy mice, PDE3B mRNA and its corresponding protein were reduced to 48 and 43%, respectively, of those in C57BL/6J control mice. Basal and insulin-stimulated membrane-bound PDE activities were also decreased to 50 and 36%, respectively, of those in the control rats. Pioglitazone increased both PDE3B mRNA and protein levels by 1.8-fold those in untreated KKAy mice. Basal and insulin-induced membrane-bound PDE activities were also increased by 1.6- and 2.0-fold, respectively. This study also suggested that mRNA and protein for PDE3 were correlated with changes in PDE3 activities. We found in the present study that when ACh was applied to mesenteric arteries, the cAMP level was significantly lower at 60 s than at 15 s, and this decrease was significantly greater in the diabetic state than in the control state (Fig. 4). These data are consistent with the increased expressions of PDE3, but not PDE4, mRNA, and protein levels in diabetic rats (Fig. 5). Together, the present results suggest that PDE3 activities are increased in STZ-induced diabetic rats. In line with this, we found that, although the ACh-induced EDHF-type relaxation was impaired in diabetic rats, the cilostamide-induced enhancement of EDHF-type relaxation was greater in diabetic rats than in control rats (Fig. 3). On the other hand, our data suggest that PDE4 may not contribute to the present EDHF-type relaxation because Ro 20–1724 was without effect.
In conclusion, we found 1) that cAMP and gap junctional communication play important roles in EDHF-type relaxation in rat mesenteric artery and 2) that the impaired EDHF-type relaxation in the mesenteric artery that we saw in STZ-induced diabetic rats may be attributed to a reduced action of cAMP, in turn resulting from increased PDE3 activity. We believe that our findings should stimulate further interest in PDE3 as a potential therapeutic target in the continuing efforts to reduce diabetes-associated vascular disease.
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ACKNOWLEDGMENTS |
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This study was supported in part by the Ministry of Education, Science, Sports and Culture, Japan.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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-glycyrrhetinic acid. Br J Pharmacol
125: 1-3,
1998.[Web of Science][Medline]
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6–9 experiments. ***P
< 0.001, **P < 0.01, L-NNA + indomethacin
group vs. L-NNA + indomethacin + 18
